Glycosyltransferases (GTases) are enzymes that post-translationally transfer glycosyl residues from an activated nucleotide sugar to monomeric and polymeric acceptor molecules such as other sugars, proteins, lipids and other organic substrates. These glucosylated molecules take part in diverse metabolic pathways and processes. The transfer of a glucosyl moiety can alter the acceptor's bioactivity, solubility and transport properties within the cell and throughout the plant. One family of GTases in higher plants is defined by the presence of a C-terminal consensus sequence. The GTases of this family function in the cytosol of plant cells and catalyse the transfer of glucose to small molecular weight substrates, such as phenylpropanoid derivatives, coumarins, flavonoids, other secondary metabolites and molecules known to act as plant hormones. Available evidence indicates that GTases enzymes can be highly specific, such as the maize and Arabidopsis GTases that glucosylate indole-3-acetic acid (IAA). We have also identified a family of Gtases which modifies abscisic acid, see WO03/023035.
Further examples of glycosyltransferases are described in the present application.
Cytokinins are plant hormones that have a structure which resembles adenine and which have various functions that include the stimulation of cell division. Cytokinins are found in most higher plants and in mosses, fungi, bacteria and in the transfer RNA of many eukaryotes and prokaryotes. There are over 200 different cytokinin compounds and are found in high concentrations in meristemic regions and areas of continuous growth, for example, roots, young leaves, developing fruits and seeds. The first cytokinin to be isolated from a plant source was called zeatin. In addition to stimulating cell division, cytokinins also stimulate morphogenesis in tissue culture, growth of lateral buds and leaf expansion as a result of cell growth. In some species cytokinins are involved in enhancing stomatal opening and promotes the conversion of etioplasts into chloroplasts by stimulation of chlorophyll synthesis. It is known that cytokinins can be modified by glucosylation.
GTases also have utility with respect to the modification of antioxidants. Reactive oxygen species are produced in all aerobic organisms during respiration and normally exist in a cell in balance with biochemical anti-oxidants. Environmental challenges, such as by pollutants, oxidants; toxicants, heavy metals and so on, can lead to excess reactive oxygen species which perturb the cellular redox balance, potentially leading to wide-ranging pathological conditions. In animals and humans, cardiovascular diseases, cancers, inflammatory and degenerative disorders are linked to events arising from oxidative damage. Because of the current prevalence of these diseases, there is considerable interest in anti-oxidants, consumed in the diet or applied topically such as in UV-screens.
Anti-oxidant micronutrients obtained from vegetables and fruits, teas, herbs and medicinal plants are thought to provide significant protection against health problems arising from oxidative stress. Well known anti-oxidants from plant tissues include for example: quercetin, luteolin, and the catechin, epicatechin and cyanidin groups of compounds. Caffeic acid (3,4-dihydroxycinnamic acid) is a further example of an anti-oxidant with beneficial therapeutic properties. Certain plant species, organs and tissues are known to have relatively high levels of one or more compounds with anti-oxidant activity. Greater accumulation of these compounds in those species, their wider distribution in crop plants, and plant parts already used for food and drink production, and the increased bioavailability of anti-oxidants (absorption, metabolic conversions and excretion rate) are three features considered to be highly desirable.
Bioreactors, for example fermentors, are vessels that comprise cells or enzymes and typically are used for the production of molecules on an industrial scale. The molecules can be recombinant proteins (e.g. enzymes such as proteases, lipases, amylases, nucleases, antibodies) or compounds that are produced by the cells contained in the vessel or via enzyme reactions that are completed in the reaction vessel. Typically, cell based bioreactors comprise the cells of interest and include all the nutrients and/or co-factors necessary to carry out the reactions.
We disclose in the present application GTases that modify compounds (e.g. cytokinins, quercetin) in a stereospecific fashion (i.e. a family of GTases which can label cytokinins or quercetin with a high degree of specificity). Moreover we disclose an in vitro cell based bioreactor that utilises these glycosyltransferases to add glucosyl moieties to these compounds.
Surprisingly we find that the bioreactor does not require an exogenous supply of UDP-glucose, (a substrate for these enzymes). We show that this is a general principle for plant GTases and applicable to any cell based bioreactor comprising transgenic cells that have been transformed with plant GTases. In our co-pending applications WO01/59140, WO02/103022, WO 03/023035 we disclose GTase sequences with substrate specificity for a range of small molecules, these sequences are specifically incorporated by reference into the current application with respect to the bioreactor embodiment.